In recent years, polyoxyethylene derivatives have very frequently used as materials for imparting a circulation in blood, a targeting function and the like toward a target site to polypeptides, enzymes, antibodies and genes, nucleic acid compounds including oligonucleic acids, nucleic acid drugs, and other physiologically active substances. This is because the derivatives exhibit a low interaction with other biological components owing to a steric repulsion effect thereof. A physiologically active substance modified with the polyoxyethylene derivative or a drug carrier obtained by modifying liposome or the like with the polyoxyethylene derivative is known to exhibit a longer circulation in blood in the body than that of unmodified one. Moreover, it has been reported that this effect increases as the molecular weight of the polyoxyethylene derivative increases. Further, it is also possible to impart the targeting function by attaching an active group or an antibody to an end of the polyoxyethylene derivative. Thus, the polyoxyethylene derivatives become very useful and indispensable materials in the field of drug delivery system. Of these, a polyoxyethylene derivative having a carboxyl group is a very important starting material as an intermediate of various active ester groups. From the viewpoint of performance and safety of drugs produced by using the derivative, as the polyoxyethylene derivative having a carboxyl group, one having a high purity and containing less amount of impurities has been desired. At present, as the polyoxyethylene derivatives having a carboxyl group, those having various skeletons or other functional groups have been developed, and impurities produced as by-products are diversified depending on production methods of the derivatives. The following will show examples of the impurities.
A polyoxyethylene derivative having a carboxyl group can be generally synthesized by using a polyoxyethylene derivative having a hydroxyl group as a starting material and oxidizing the end hydroxyl group into a carboxyl group. Usually, in the case of an end modification reaction of a polyoxyethylene derivative, since the reaction occurs at a polymer end, concentration of the reaction site in the molecule decreases and thus reaction conversion gets worse, so that the hydroxyl group may remain.
Moreover, as the molecular weight increases, viscosity of the reaction solution increases and reaction conversion gets worse, so that unreacted hydroxyl group remains. In the case of a multi-arm type polyoxyethylene derivative having a large number of hydroxyl groups, impurities having different numbers of unreacted hydroxyl groups form at the end modification. In general, since the derivative having an end hydroxyl group and the derivative having an end carboxyl group are similarly polyoxyethylene derivatives, purification is difficult. Particularly, as the molecular weight increases, the physical properties thereof become more resemble and hence separation and purification are difficult. Moreover, in the case of the polyoxyethylene derivative in which such an end unreacted hydroxyl group remains, dimerization proceeds in the following active ester formation and hence purity of the active ester compound is remarkably decreased.
In addition, in the case where impurities such as the derivative having a hydroxyl group or the dimmer have remained, the impurities remains as polymer impurities also at a drug modification reaction in a later step, so that the resulting drug becomes inhomogeneous and is problematic as a pharmaceutical preparation.
As a purification method of the polyoxyethylene derivative having a carboxyl group at an end, a column chromatographic purification using an ion-exchange resin shown below is known (Patent Document 1 (U.S. Pat. No. 5,298,410): Patent Document 2 (JP-T-2008-514693)). In these Patent Documents, for the purpose of removing a diol compound contained in methoxypolyethylene glycol, there is disclosed a method of modifying the hydroxyl group into a carboxyl group and subsequently separating and fractionating a monocarboxyl compound and a dicarboxyl compound by ion-exchange chromatography. As a specific purification method, after an end carboxyl group-containing methoxypolyethylene glycol derivative (molecular weight: 20,000) is dissolved in distilled water to form an approximately 2% aqueous solution and the solution is charged onto an ion-exchange resin, distilled water is introduced thereto to separate the impurity from the objective material. The resulting fractionated fraction is further diluted to form an approximately 1% aqueous solution. After pH adjustment, purification is achieved by performing extraction with dichloromethane, dehydration, and crystallization. However, in the case of column chromatographic purification using an ion-exchange resin as described above, a sample should be a highly diluted one (an aqueous solution having a PEG concentration of 1 to 2%). This is because the objective material is a carboxyl compound only at a polymer end and exhibits an extremely low interaction with the ion-exchange resin. Moreover, in order to remove water from the dilute aqueous solution after fractionation to obtain the objective material, a large-volume extraction apparatus is needed.
In addition, efficiency is poor also from the consideration of the required time from the purification until the recovery of the objective material, so that the purification by the ion-exchange chromatography is problematic as an industrial production method.
As another example of the polyoxyethylene derivative having a carboxyl group, there is a method of synthesizing the derivative by chemically bonding a methoxypolyoxyethylene derivative to an amino group or a thiol group of a compound containing a carboxyl group, such as an amino acid.
As a specific example, Patent Document 3 (U.S. Pat. No. 5,932,462) describes a synthetic method of a polyoxyethylene derivative (molecular weight: 40,000) where two chains of a methoxypolyoxyethylene derivative (molecular weight: 20,000) are introduced into two amino groups of lysine and a purification method thereof. The impurities in this case include the unreacted methoxypolyoxyethylene derivative having a molecular weight of 20,000, one (molecular weight: 20,000) where one chain thereof is introduced into lysine, further one (molecular weight: 40,000 or more) where a diol compound (molecular weight: 40,000) that is an impurity in the methoxypolyoxyethylene derivative (molecular weight: 20,000) is introduced into lysine, one (molecular weight: 40,000 or more) where three chains of the polyoxyethylene derivative are introduced into a dimer that is an impurity in lysine, and the like.
As a purification method of the impurities as described above, Patent Document 3 (U.S. Pat. No. 5,932,462) proposes column chromatography on an ion-exchange resin. Specifically, an aqueous solution adjusted so that concentration of the polyoxyethylene derivative becomes 0.2% is charged into a column of the ion-exchange resin and fractionation is performed using ion-exchange water as an eluent. On this occasion, the impurities having a molecular weight of 20,000 are eluted faster than the objective material and then the objective material is eluted. After the objective material, there are eluted the impurities having a molecular weight of 40,000 or more and the impurity where one chain of the methoxypolyoxyethylene derivative is introduced into a lysine having a molecular weight of 20,000. In order to fractionate the impurities and the objective material, it is necessary to collect aqueous solutions containing the objective material alone through fine fractionation and analysis of eluting solutions during purification. In addition, this method is a method of separating a high-molecular-weight polyoxyethylene derivative and hence it is necessary to dilute a sample to be charged into a column to a degree more than the degree in the method described above (solution concentration: 0.2%). Moreover, since the purification is performed in an aqueous solution, steps of extraction, dehydration, concentration, and crystallization are subsequently necessary and thus operations are extremely vexatious and complex. When required time and apparatus to be used for the amount to be purified and the treatment of the distilled water and organic solvent used for the purification are considered, the purification by the column chromatography is industrially disadvantageous as in the case of the method described above.